PROJECT SUMMARY Accumulating evidence suggest that neurodegenerative diseases including Alzheimer?s disease (AD) are frequently the result of alterations in endosomal trafficking and proteostasis pathways, one consequence of which can be the accumulation of aggregation-prone proteins. Numerous familial and sporadic loss or gain-of- function mutations have been identified in such pathways, illuminating potential drivers of disease pathogenesis. In addition, excess protein mis-folding due to altered trafficking could affect proteostasis pathways by blocking protein turnover or trafficking. Protein and organelle trafficking within cells is a highly dynamic and interconnected process, and defects in one arm of the system can affect other aspects of the network in unpredictable ways including reduced flux in turnover pathways. Indeed, it is conceivable that many seemingly unrelated mutations across the trafficking landscape in various neurodegenerative diseases reveal common mechanistic vulnerabilities downstream but with distinct cell-type sensitivities reflective of the identity of mis-trafficked proteins. As such, understanding the global architecture of trafficking systems and the key machinery that controls the directionality and efficiency of trafficking, particularly of aggregation-prone neurodegenerative proteins such as APP and its aggregation-prone form A?, represents a central goal of the field. A? aggregation as a toxic driver of AD neuropathology has been a dominant hypothesis in the field. However, thus far therapeutics directed at aggregate prevention or removal have not been successful, and alternative hypotheses including alterations in intracellular trafficking as an important event in neuropathology have emerged. Here, we seek to combine powerful genetic and proteomic approaches to develop a quantitative framework for understanding how disruption of major endosomal trafficking systems ? retromer and retriever, found defective in neurodegenerative diseases ? alter global membrane protein trafficking, and specific trafficking and processing of APP proteoforms. These studies make use of an extensive tool-kit of mutant tissue culture cell lines and induced neurons derived from human embryonic stem cell (hESC), in combination with targeted and unbiased proteomics of individual organelles linked with endosomal trafficking, to assemble a global map of cargo and trafficking dependencies. In parallel, we will employ novel flux-based screening strategies to search for genes controlling APP/A? trafficking to the lysosome and the plasma membrane, and will examine the extent to which A? accumulation within the endo-lysosomal system alters selective autophagic flux using new cargo-specific reporters. The central hypothesis being tested is that specific defects in protein trafficking networks underlies the susceptibility of neurons to A? and other aggregation prone proteins and that these defects can be molecularly unmasked through systematic network and genetic analysis.